Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
  • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
  • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
  • C07K16/1036—Retroviridae, e.g. leukemia viruses
  • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/01—Animal expressing industrially exogenous proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• Methods and compositions for immunogen design using B cell lineage design in non-human animals humanized immunoglobulin loci Methods and compositions for developing immunogens for making vaccines, employing non-human animals with humanized immunoglobulin loci. Iterative processes of priming and boosting of non- human animals having humanized immunoglobulin loci to identify candidate immunogens for use as vaccines and for generating broadly neutralizing antibodies against infectious agents and pathogens. Development of human vaccines in humanized immunoglobulin animals.
• Vaccines against human pathogens are sorely needed, but difficult to obtain. Few, if any, suitable methods or systems are available for exposing a human immune system to a human infectious agent or pathogen and obtaining useful information that can be used to design a suitable vaccine that induces immunity in a human subject to the infectious agent or pathogen of interest. It is often not ethical, nor practical, to expose human subjects to immunogens derived from a pathogen or an infectious agent in a protocol for designing a vaccine, and non-human animal systems are typically unsuitable for obtaining useful information about human immunogenicity.
• the process involves a search for a key immunogen that can prime the naive human immunoglobulin repertoire and, with judiciously selected boost immunogens, drive the B cell clonal selection process to produce a mature B cell that expresses broadly neutralizing antibodies.
• compositions and methods for generating vaccines that are suitable for immunizing humans against human infectious agents.
• rational methods for designing immunogens to be used as vaccines in human subjects There is a need in the art for humanized systems, e.g., humanized animals, that can be used in methods to generate vaccines and vaccine candidates to immunize humans against human infectious agents.
• humanized systems e.g., humanized animals
• a non-human platform that can recapitulate a human B cell response, for making vaccines to infectious agents by promoting development of desirable B cells through the use of immunogen variants that will ultimately result in the development of desirable B cells that express broadly neutralizing antibodies against an infectious agent of interest.
• humanized animals to further develop broadly neutralizing antibodies to epitopes of infectious agents.
• compositions and methods are provided for vaccine design using non- human animals comprising humanized immunoglobulin sequences, including iterative processes that comprise employing selected immunogens to develop B cell clones from the naive repertoire ⁇ e.g, immature B cells comprising early B cell receptors) that mature into B cells that produce useful antibodies that selectively bind particular immunogens, wherein one or more of the selected immunogens are employed as vaccines.
• compositions and methods are described for generating immune cells (in genetically modified non-human animals) that bind particular immunogens of interest, including developing immune cells from naive or non- mutated states to mature or hypermutated states that specifically bind an infectious agent of interest, and employing one or more immunogens of interest which achieved those steps in a vaccine preparation to develop in a subject in need thereof an immune response to an infectious agent of interest, e.g., to develop an immunity in the subject of interest to the infectious agent of interest, and immune cell populations that express immunoglobulin sequences that bind antigens of interest.
• Methods for making vaccines using rodents with humanized immunoglobulin sequences are provided.
• compositions and methods are provided for facilitating B cell lineage immunogen design using non-human animals having humanized immunoglobulin loci.
• Compositions and methods are provided for using the non-human animals to identify, through intermediate ancestor B cells and B cells bearing unmutated receptors (e.g., a naive B cell repertoire, or a B cell repertoire characterized by germline rearrangements that have not been mutated, or have not been somatically hypermutated), immunogens to develop vaccines against infectious agents, which will elicit production from a human immunoglobulin repertoire of mature B cells that express broadly neutralizing antibodies.
• unmutated receptors e.g., a naive B cell repertoire, or a B cell repertoire characterized by germline rearrangements that have not been mutated, or have not been somatically hypermutated
• compositions and methods are provided for prime and boost cycles using variants of immunogens and, optionally, adjuvants that generate broadly neutralizing antibodies, such that immunogen variants capable of stimulating an early or naive human B cell repertoire (e.g., a repertoire reflecting germline rearrangements) are identified, wherein the immunogen variants induce the production of a broadly neutralizing antibody against the infectious immunogen.
• immunogen variants capable of stimulating an early or naive human B cell repertoire (e.g., a repertoire reflecting germline rearrangements) are identified, wherein the immunogen variants induce the production of a broadly neutralizing antibody against the infectious immunogen.
• compositions and methods for using non-human animals having restricted immunoglobulin repertoires are also provided.
• methods and compositions are provided using B Cell Lineage-based immunogen design in non-human animals comprising a humanized immunoglobulin locus (e.g. , the VELOCIMMUNE® humanized mouse) for iterative development of vaccine immunogens for infectious diseases.
• a humanized immunoglobulin locus e.g. , the VELOCIMMUNE® humanized mouse
• infectious disease agent components are used as antigen-specific labels to identify precursors of protective antibodies in unimmunized or uninfected VELOCIMMUNE® humanized mice or using similar reagents and related strategies to identify immunogen-induced clonal lineages of anti-infectious agent antibodies that are developing in the desired direction. These antibodies are then made to use as templates for iterative vaccine design, wherein a series of novel immunogens and, optionally, adjuvants to drive an otherwise subdominant or disfavored B cell maturation pathway to become dominant is determined. In one embodiment, sufficient plasma and tissue levels of antibody are made to be protective in the setting of vaccination.
• non-human animals comprising humanized immunoglobulin loci (e.g. , VELOCIMMUNE® humanized mice) are used to design of vaccine immunogens comprising: employing pre-immune unvaccinated mouse bone marrow to isolate antigen-specific naive B cells that bind to antigen-specific infectious agent reagents bearing broadly neutralizing epitopes; the antibodies reflective of the naive B Cell Receptors (BCR) are isolated and made recombinantly; and immunogens are selected for their high affinity binding to these BCR in order to discover immunogens that would selectively drive desired broadly neutralizing B cell lineages to mature in peripheral lymph nodes and spleen.
• BCR naive B Cell Receptors
• non-human animals comprising humanized immunoglobulin loci are vaccinated with existing immunogens that themselves are antigenic for broadly neutralizing antibodies one is trying to induce; the antibodies induced are then isolated by making monoclonal antibodies from the spleens or lymph nodes of the immunized mice; the antibodies are made recombinantly; and then the most mature antibodies in the desired lineage used as a template to further drive the desired lineage to full expression of the desired function, e.g., a protective effector function against an infectious agent.
• the protective function includes virus neutralization, antibody-dependent cellular cytotoxicity against infectious agent- infected cells, prevention of movement of infectious agent across mucosal barriers, and blocking of entry of infectious agent in cell targets.
• a method for obtaining an immunogen that results in production in a non-human animal comprising a humanized Ig locus of a broadly neutralizing antibody against an infectious agent comprising iterating and screening candidate immunogens for B cell lineage vaccine design, wherein the iterating and screening comprises:
• the method further comprises designing a fourth epitope that specifically binds a third intermediate ancestor antibody of the broadly neutralizing antibody, wherein the fourth epitope is employed as a vaccine against the infectious agent.
• the method further comprises designing a fifth epitope that specifically binds a fourth intermediate ancestor antibody of the broadly neutralizing antibody, wherein the fifth epitope is employed as a vaccine against the infectious agent.
• the first, second, third, fourth, or fifth epitope binds an unmutated ancestor antibody.
• the first, second, third, fourth, or fifth epitope that binds the unmutated ancestor antibody is employed as a vaccine against the infectious agent.
• the unmutated ancestor antibody is an antibody of a naive B cell. In one embodiment, the unmutated ancestor antibody is displayed on an early B cell as an IgM antibody. In one embodiment, the unmutated ancestor antibody comprises a rearrangement of germline gene segments prior to class switching.
• a method for making a vaccine against an infectious agent in a non-human animal that comprises a humanized immunoglobulin locus comprising priming and boosting with variants of an epitope of the infectious agent, wherein the epitope of the infectious agent binds to a broadly neutralizing antibody.
• the priming and boosting with variants of the epitope of the infectious agent is continued until a variant is found that, upon immunizing a naive non-human animal with a humanized Ig locus, the variant produces a broadly neutralizing antibody.
• the variant that produces a broadly neutralizing antibody is employed as a vaccine against the infectious agent.
• a method for designing a broadly neutralizing antibody (BNAb) in a non-human animal comprising a humanized immunoglobulin locus comprising exposing the animal to a first structural variant of an epitope of an immunogen that binds a broadly neutralizing antibody, allowing the animal to develop a first immune response to the first structural variant, and screening antibodies in the animal to obtain a broadly neutralizing antibody against the immunogen.
• BNAb broadly neutralizing antibody
• the method comprises exposing exposing the animal to a second structural variant of the epitope that binds the broadly neutralizing antibody, allowing the animal to develop a second immune response, and screening antibodies from the animal to obtain a broadly neutralizing antibody against the immunogen, wherein the immunogen, the first structural variant, and the second structural variant are not identical.
• a method for employing a non-human animal comprising a humanized immunoglobulin locus in an algorithm to determine an unmutated ancestor antibody, comprising administering to the non-human animal a first variant of an immunogen that binds a broadly neutralizing antibody.
• the algorithm employs quantitative inference based on the number of different VDJ combinations (roughly 10 9 ) compared with the number of possible nucleotide sequences of comparable length (roughly 4 350 ). The algorithm infers back from the sequence of the broadly neutralizing antibody to the most plausible ancestor, or ancestors.
• the unmutated ancestral VDJ is determined by modeling the somatic mutation rates, the probability that a given nucleotide will be in a mutated states, and the number of passages through somatic hypermutation. See, e.g., Haynes et al. (2012) B-cell-lineage immunogen design in vaccine development with HIV-1 as a case study, Nature Biotech. 30(5):423-433 (hereby incorporated by reference, see, e.g., especially Box 4).
• a method for employing a non-human animal with a humanized immunoglobulin locus to optimize and validate immunogens to develop a vaccine against an infectious agent comprising priming and boosting the non-human animal with an immunogen of interest for B cell lineage vaccine design.
• the non-human animal is primed and boosted with a first pre-selected immunogen, a second pre-selected immunogen, and an nth preselected immunogen, until the non-human animal expresses a broadly neutralizing antibody against the infections agent.
• the first pre-selected immunogen, the second pre-selected immunogen, or the nth pre-selected immunogen is an effective vaccine against the infectious agent.
• nth refers to third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth.
• a method for making a vaccine that elicits production in a human subject of a broadly neutralizing antibody against an infectious agent of interest comprising priming and boosting a non-human animal comprising a humanized Ig locus with variants of an epitope of the infectious agent that binds a broadly neutralizing antibody, wherein a priming and boosting process employing variants of the epitope of the infectious agent that do not bind the broadly neutralizing antibody, but produce in the non-human animal a broadly neutralizing antibody that binds the infectious agent of interest.
• a method for making a vaccine that elicits in a human a broadly neutralizing antibody against an infectious agent of interest comprising identifying an antibody that broadly neutralizes the infectious agent of interest, making variants of the epitope bound by the broadly neutralizing antibody, performing prime and boost cycles with variants of the epitope bound by the broadly neutralizing antibody until an effective variant results in production of an antibody that broadly neutralizes the infectious agent of interest, and employing the effective variant as a vaccine against the infectious agent of interest.
• a method for B cell lineage-based vaccine design comprising: (a) immunizing a non-human animal that comprises a humanized immunoglobulin locus with a first immunogen of interest, wherein the first immunogen of interest comprises a plurality of epitopes; (b) allowing the non-human animal to mount an immune response to the first immunogen of interest, wherein the immune response comprises creating non-human B cells that express human immunoglobulin heavy chain VDJ and human immunoglobulin light chain VJ domains; (c) isolating clonally related memory B cells from the non-human animal that specifically bind a first epitope of interest on the first immunogen of interest; (d) determining VDJ and VJ sequence information for the VDJ and VJ sequences of the memory B cells; (e) employing the determined VDJ and VJ sequence information to determine an unmutated B cell receptor (BCR) VDJ and VJ sequence and, optionally, one or more intermediate ancestor
• the first immunogen of interest is, or is derived from, an infectious agent or a pathogen.
• the infectious agent or pathogen is a virus, a bacterium, a fungus, or a parasite.
• the infectious agent is selected from HIV-1 , hepatitis C, dengue fever virus, and HPV.
• the first immunogen of interest is a protein or a peptide and the second immunogen of interest is a structural variant of the first immunogen of interest.
• the structural variant comprises a substitution of one or more amino acids of the protein or peptide.
• the first immunogen is a protein with a postranslational modification (e.g., glycosylation, or presence of a non-protein group, e.g., a lipid), and the second immunogen of interest is a variant of the postranslational modification.
• the VDJ is derived from a human V H 1-69 gene segment and a human D and a human J segment,
• a method for designing a vaccine against an infectious agent or pathogen comprising employing a non-human animal that comprises a humanized immunoglobulin iocus as a
• broadly neutralizing antibodies of interest are antibodies that exist in human subjects who are infected with an infectious agent of interest, wherein the antibodies are capable of neutralizing the infectious agent in culture.
• the antibodies are capable of preventing infection of a cell that said infectious agents can normally infect in the absence of the antibodies.
• the infectious agent or pathogen is selected from a virus ⁇ e.g., human immunodeficiency virus (HIV), e.g., HIV-1 , an influenza virus strain, human papillomavirus (HPV), hepatitis C, hepatitis B, hepatitis A, dengue), a bacterium, a fungus, a parasite (e.g. , a malaria strain or species), etc.
• HIV human immunodeficiency virus
• HPV human papillomavirus
• HPV human papillomavirus
• hepatitis C hepatitis B
• hepatitis A dengue
• dengue a virus
• bacterium e.g. , a fungus
• a parasite e.g. , a malaria strain or species
• a non-human animal comprising a humanized immunoglobulin Iocus
• the animal is immunized with a variant of an epitope of an infectious agent to which a first broadly neutralizing human antibody binds
• the animal as the result of the immunization with the epitope variant comprises a second broadly neutralizing antibody that broadly neutralizes the same epitope of the same infectious agent, wherein the second broadly neutralizing antibody is not identical to the first broadly neutralizing antibody.
• compositions and methods are provided for identifying immunogens for use as vaccines against infectious agents, and for developing broadly neutralizing antibodies against infectious agents, using non-human animals comprising a humanized immunoglobulin Iocus that express a B cell population derived from the entire human heavy chain variable gene repertoire, operably linked to a plurality of human D segments and a plurality of human J segments; wherein the variable segments are operably linked to a constant region, e.g., a human or non-human constant region on a transgene, or an endogenous non-human constant region operably linked at the endogenous non-human Iocus to the human heavy chain repertoire.
• a constant region e.g., a human or non-human constant region on a transgene, or an endogenous non-human constant region operably linked at the endogenous non-human Iocus to the human heavy chain repertoire.
• the non-human animal comprises a full and unrearranged human ⁇ and/or ⁇ light chain variable repertoire, on a transgene linked to a human or rodent (e.g. , mouse or rat) constant region gene, or at an endogenous non-human light chain locus operably linked to a rodent (e.g., mouse or rat) constant region gene.
• a human or rodent e.g. , mouse or rat
• rodent e.g., mouse or rat
• compositions and methods are provided for identifying immunogens for use as vaccines against infectious agents, and for developing broadly neutralizing antibodies against infectious agents, using non-human animals comprising a humanized immunoglobulin locus that express a B cell population derived from no more than one, or no more than two, heavy chain variable region family members (e.g., V H 1 -69 and/or its polymorphs; V H 1 -2 and/or its polymorphs) operably linked to a plurality of human D segments and a plurality of human J segments; wherein the variable segments are operably linked to a constant region, e.g., a human or non- human constant region on a transgene, or an endogenous non-human constant region operably linked at the endogenous non-human locus to the restricted human V H repertoire.
• V H 1 -69 and/or its polymorphs V H 1 -2 and/or its polymorphs
• the non-human animal comprises a full and unrearranged human ⁇ and/or ⁇ light chain variable repertoire, on a transgene linked to a human or rodent (e.g. , mouse or rat) constant region gene, or at an endogenous non-human light chain locus operably linked to a rodent (e.g., mouse or rat) constant region gene.
• a human or rodent e.g. , mouse or rat
• rodent e.g., mouse or rat
• non-human animals are provided that are immunized with human infectious agents, wherein the non-human animals comprise unrearranged human immunoglobulin variable region gene segments that are capable of rearranging and forming genes that encode variable region sequences operably linked to endogenous non-human constant regions, wherein the variable domains encoded by the sequences specifically bind the infectious agent.
• the non-human animal is immunized with an immunogen that is a variant of an immunogen that binds a broadly neutralizing human antibody, e.g., a broadly neutralizing antibody isolated from a patient infected with the infectious agent).
• the immunogen that the non-human animal is immunized with is not identical to the immunogen that binds the broadly neutralizing antibody.
• a non-human animal with a humanized immunoglobulin heavy chain variable region gene locus and a humanized light chain variable region gene locus is employed to screen for (or use in prime and boost cycles) immunogens to employ as vaccines against human infectious agents or pathogens.
• the immunogens are derived from, but are not identical to, immunogens that bind broadly neutralizing antibodies against the infectious agent or pathogen.
• the identity of the screening immunogen is about 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% identical to the immunogen that binds the broadly neutralizing antibody.
• the non-human animals comprising a humanized immunoglobulin locus comprises, e.g., an insertion of one or more unrearranged human heavy chain V, D, and J and/or unrearranged human light chain V and J gene segments, either on a transgene (fully human or human variable/non-human constant) or operably linked to a non-human constant region at an endogenous non- human locus.
• the genetically modified heavy chain locus comprises an endogenous Adam6a gene, Adam6b gene, or both, and the genetic modification does not affect the expression and/or function of the endogenous Adam6a gene, Adam6b gene, or both.
• the non-human animal comprises an ectopically present Adam6a gene, Adam6b gene, or both.
• the Adam6a gene is a non-human Adam6a gene.
• the Adam6a gene is a mouse Adam6a gene.
• the Adam6a gene is a human
• the Adam6a gene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is a mouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6b gene.
• the one or more unrearranged human heavy chain V, D, and J and/or unrearranged human light chain V and J gene segments are present in a restricted repertoire, e.g. , the number of human heavy and/or light gene segments present in the non-human animal is less than a complete human repertoire of the corresponding gene segments.
• the human gene segments are inserted at an endogenous locus (directly or through a recombinase-containing cassette), and endogenous unrearranged gene segments are retained in the genome of the non-human animal.
• the human gene segments are inserted between the 3'-most non-human gene segment and the first non-human constant gene.
• endogenous non-human variable gene segments are present, they are inactivated, e.g., by an inversion or a partial deletion.
• the non-human animal is a rodent, e.g., a mouse or a rat.
• the non-human animal comprises a plurality of unrearranged human light chain V and J gene segments operably linked to a constant region (e.g., a human or non-human constant region, at an endogenous Ig locus or on a transgene), and the unrearranged human heavy chain locus is restricted to no more than one, or no more than two, or no more than three V H gene segments operably linked to one or more human D segments and one or more human J segments, at an endogenous Ig locus or on a transgene.
• a constant region e.g., a human or non-human constant region, at an endogenous Ig locus or on a transgene
• the non-human animal is a mouse or rat that comprises a germline modification resulting in no more than one, no more than two, or no more than three human V H gene segments operably linked to a plurality of human D and a plurality of human J gene segments, wherein the mouse or rat comprises a plurality of unrearranged human VK and JK and/or human V and ⁇ gene segments operably linked to a human or a non-human constant region gene.
• the plurality of unrearranged human VK and JK and/or human ⁇ and ⁇ gene segments are operably to endogenous constant region genes at an endogenous non-human light chain locus.
• a method for obtaining a broad spectrum antibody against an antigen of interest comprising:
• a genetically modified immunoglobulin heavy chain locus comprising one or more human V H gene segments, a plurality of human D H gene segments, and a plurality of human J segments, wherein the VH, D h , and J H gene segments are operably linked to a heavy chain constant region nucleic acid sequence
• a genetically modified immunoglobulin light chain locus comprising one or more human VK gene segments and one or more human JK gene segments, wherein the VK and the JK gene segments are operabiy linked to a light chain constant region nucleic acid sequence, and
• the first immunogen comprises a plurality of epitopes
• the immune response comprises generation of non-human B cells that express human immunoglobulin heavy chain (IgH) VDJ and human immunoglobulin light chain (IgL) VJ sequences;
• the immune response of the non-human animal is boosted by serially administering the plurality of immunogens identified in step (e) until the broad spectrum antibody is produced by the non-human animal.
• the immune response of the non-human animal is boosted by administering a combination of the plurality of the immunogens identified in step (e).
• the antigen of interest is, or is derived from, an infectious agent or a pathogen
• the broad spectrum antibody is a broadly neutralizing antibody against the infectious agent or the pathogen.
• the infectious agent or pathogen is selected from the group consisting of a virus, a bacterium, a fungus, and a parasite.
• the infectious agent is selected from the group consisting of a human immunodeficiency virus (HIV), a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a dengue fever virus, and a human papillomavirus (HPV).
• HAV human immunodeficiency virus
• HPV human papillomavirus
• all, or substantially all, functional endogenous V H D H , and J H gene segments in the immunoglobulin heavy chain locus have been deleted or rendered non-functional, and wherein the genetically modified immunoglobulin heavy chain locus comprises a single human V H gene segment or a polymorphic variant thereof, one or more human D H gene segment, and one or more human J H gene segment.
• the genetically modified heavy chain locus comprises an endogenous Adam6a gene, Adam6b gene, or both, and the genetic modification in the heavy chain locus does not affect the expression or function of the endogenous Adam6a gene, Adam6b gene, or both.
• the non-human animal comprises an ectopically present Adam6a gene, Adam6b gene, or both.
• the single human V H gene segment is selected from the group consisting of V H 1-2, V H 1-3, V H 1-8, V H 1-18, V H 1-24, V H 1-45, V H 1-46, V H 1-58, V H 1-69, V H 2-5, V H 2-26, V H 2-70, V H 3-7, V H 3-9, V H 3-11 , V H 3-13, V H 3-15, V H 3- 16, V H 3-20, V H 3-21 , V H 3-23, V H 3-30, V H 3-30-3, V H 3-30-5, V H 3-33, V H 3-35, V H 3-38, V H 3-43, V H 3-48, V H 3-49, V H 3-53, V H 3-64, V H 3-66, V H 3-72, V H 3-73, V H 3-74, V H 4-4, V H 4-28, V H 4-30-1 , V H 4-30-2, V H 4-30-4, V H 4-31 , V H 4-34, V H 4-39, V H 4-59
• the single human V H gene segment is a human V H 1-69 gene segment or a polymorphic variant thereof.
• the single human V H gene segment is a human V H 1-2 gene segments or a polymorphic variant thereof.
• the single human V H gene segment is a human V H 4-59 gene segment or a polymorphic variant thereof.
• the non-human animal is a rodent.
• the non-human animal is a mouse or a rat.
• the constant region nucleic acid sequence is a human or a rodent nucleic acid sequence.
• the non-human animal is a mouse and the constant region nucleic acid sequence is a mouse constant region nucleic acid sequence.
• all, or substantially all, functional endogenous V H D H , and J H gene segments in the immunoglobulin heavy chain locus have been rendered non-functional, and the human V H , DH, and JH gene segments are present on a transgene.
• the human V H , D H , and J H gene segments are operably linked to an endogenous constant region nucleic acid sequence at an endogenous non-human immunoglobulin heavy chain locus.
• the genetically modified immunoglobulin light chain locus comprises a replacement of all, or substantially all, functional VK gene segments, JK gene segments with one or more human VK gene segments and one or more JK gene segments, wherein the human VK gene segments and the JK gene segments are operably linked to a light chain constant region nucleic acid sequence.
• the non-human animal comprises a replacement at the endogenous non-human immunoglobulin VK locus of all or substantially all functional endogenous VK gene segments with human VK gene segments selected from VK4-1 , VK5-2, VK7-3, VK2-4, VK1-5, VK1 -6, VK3-7, VK1-8, VK1 -9, VK2-1 0, VK3- 1 1 , VK1-12, VK1-13, VK2-14, VK3-15, VK1-16, VK1-17, VK2-18, VK2-19, VK3-20, VK6- 21 , VK1 -22, VK1-23, VK2-24, VK3-25, VK2-26, VK1-27, VK2-28, VK2-29, VK2-30, VK3- 31 , VK1 -32, VK1-33, VK3-34, VK1 -35, VK2-36, VK1-37, VK2-38, VK1-39,
• the non-human animal comprises a replacement at the endogenous non-human immunoglobulin JK locus of all or substantially all functional endogenous non-human immunoglobulin JK gene segments with human JK gene segments selected from JK1 , JK2, JK3, JK4, JK5, and a combination thereof.
• the one or more human VK and JK segments are operably linked to an endogenous light chain constant region nucleic acid sequence at an endogenous non-human locus.
• FIG. 1 illustrates general steps of the B cell lineage design-based approach to vaccine design.
• the Ab1 , Ab2, Ab3, and Ab4 refer to four different antibody clonal lineages from peripheral blood memory B cells, one or more of which produces a protective antibody that is desired to be induced.
• Step 2 involves inference of an unmutated ancestor (UA) and intermediate (IA) antibodies in an antibody clonal lineage.
• Step 3 involves suing the UA(s)and lAs as templates to design immunogens with high affinity US and IA binding.
• This schematic illustrates a general scheme for regressing from an epitope of an infectious agent that binds a mature, broadly neutralizing antibody to an unmutated ancestor B cell (e.g., UA) that binds a priming immunogen.
• an unmutated ancestor B cell e.g., UA
• UA unmutated ancestor B cell
• IA an antigen for boosting
• a process that is iterated until the mature broadly neutralizing antibody is obtained.
• the iterative process allows for identification of suitable immunogens (antigens for prime and boosting) that may serve in the design of vaccines to elicit mature broadly neutralizing antibodies. See, Haynes ef a/. (2012) B-cell-lineage immunogen design in vaccine development with HIV-1 as a case study, Nature Biotech.
• FIG. 2 lists two types of VELOCIMMUNE® humanized mice that can be used for this approach.
• Top panel a VELOCIMMUNE® humanized mouse with a replacement of mouse endogenous sequences (closed symbols) with human genomic sequences (closed symbols);
• Bottom panel a V H restricted VELOCIMMUNE® humanized mouse with a single V H 1-9 gene segment replacing all endogenous mouse V H gene segments, operably linked to human D and human J gene segments; for simplicity, the humanized ⁇ locus is not shown for the Human V H 1 -69 panel.
• Other V H restricted VELOCIMMUNE® humanized mice can similarly be used.
• FIG. 3A shows normal B cell subsets of immune cells in VELOCIMMUNE® humanized mice in bone marrow, spleen, and peritoneum, as compared with wild-type BL/6 mice.
• FIG. 3B shows normal B cell subsets of immune cells in VELOCIMMUNE® humanized mice in Peyer's patch.
• FIG. 4 shows normal T cell development and myeloid cell development of immune cells in VELOCIMMUNE® humanized mice.
• Top panel BL/6 wild-type mice;
• Bottom panel a V H restricted VELOCIMMUNE® humanized mouse with a single V H 1-9 gene segment replacing all endogenous mouse V H gene segments.
• T FH B220 CF ⁇ + CD4 + CXCR5 + PD-1 + Foxp3
• T FR B220 " TCRp + CD4 + CXCR5 + PD-1 + Foxp3 + .
• FIG. 5A shows plasma anti HIV-1 Envelope responses of VELOCIMMUNE® humanized mice to immunization with the HIV Envelope AE.
• A244 gp120 protein Alam, S.M. et al. (2013) Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N- Terminal Deletion, J. Virol. 87: 1554-68).
• FIG. 5B shows plasma anti HIV-1 Envelope responses of VELOCIMMUNE® humanized mice to immunization with the gp41 membrane proximal external region (MPER) liposome antigen.
• the plotted lines from left to right, reflect Mouse RE506, Mouse RE506, Mouse RE507, and Mouse RE508.
• VELOCIMMUNE® humanized mice were immunized i.p. with MPER 656-GTH1 liposomes/R848/oCpg/ LipidA.
• FIG. 6 shows a schema of the gp41 MPER liposome-peptide immunogen (to the left) used in FIG. 5, containing the neutralizing epitopes for the HIV-1 broadly neutralizing antibodies 2F5 and 4E10 (Dennison, S.M. et al., (2009) Stable Docking of Neutralizing Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Monoclonal Antibodies 2F5 and 4E10 Is Dependent on the Membrane Immersion Depth of Their Epitope Regions, J. Virol, 83:1021 1-10223).
• FIG. 7 shows the ability to use a B cell tetramer reflective of the gp41 2F5 MPER neutralizing epitope (Verkoczy, L et al. (2009) Functional, Non-Clonal lgM a -Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region, PLoS One 4( 0): e7215, 2009) to identify and isolate naive B cell precursors of this lineage from pre-immune VELOCIMMUNE® humanized mice.
• the term "broad spectrum antibody” as used herein includes antibodies produced by B cells that can bind a plurality of epitopes or antigenic determinants of an antigen, in one embodiment, the broad spectrum antibody possesses polyreactivity against various epitopes or antigenic determinants. In one embodiment, the broad spectrum antibody includes autoreactive antibodies expressed by ancestral B cells, immatuare autoreactive B cells, or intermediate B cells.
• narrowly neutralizing antibody includes antibodies produced by B cells that neutralize diverse strains of a particular infectious agent or a pathogen.
• the unmutated ancestor antibody or "UA” as used herein includes antibodies expressed by a naive B cell.
• the unmutated ancestor antibody is displayed on an early B cell as an IgM antibody.
• the unmutated ancestor antibody comprises a rearrangement of germline gene segments prior to class switching.
• intermediate antibodies or "IA” used herein includes antibodies made by intermediates in the clonal lineage generated by affinity maturation of a naive B cell in a germinal center.
• Germinal center as used herein includes a location in immune tissues at which dendritic and other cells present B cell contact antigen, helper T cells make contact with B cells, and immunoglobulin class switching and somatic hypermutation take place.
• a number of infectious diseases are characterized as having regions of the outer coats, envelopes or virion spikes to which neutralizing antibodies can be made, but for a variety of reasons, are not routinely made.
• infections include HIV-1 , Hepatitis C, influenza and dengue (reviewed in Haynes, BF et al. (2012) B-cell-lineage immunogen design in vaccine development with HIV-1 as a case study, Nature Biotech 30:423-433).
• a strategy for overcoming many of the road-blocks in induction of such protective anti-infectious agent antibodies has been proposed termed B Cell Lineage Immunogen Design, in which clonal lineages of rare desired antibodies are isolated, recombinantly expressed, and used as templates for vaccine design.
• the VELOCIMMUNE® humanized mouse represents the most advanced form of mice that carry humanized Ig loci.
• the full complement of human V H , VK, D, J h and JK gene segments replace the endogenous loci.
• VELOCIMMUNE® humanized mice are capable of class-switch recombination, somatic hypermuation, and affinity-driven selection in germinal centers— thus they are and excellent and unique model to rapidly vaccinate or select pre-vaccination naive precursors for use with B cell lineage immunogen design to design novel immunogens for infectious agent vaccine development.
• VELOCIMMUNE® humanized mice that selectively express a limited V H repertoire, or only one human V H (such as V H 1-69 as just one representative example), can also advantageously be used for this purpose.
• the VELOCIMMUNE® humanized mice can be used in two ways for design of vaccine immunogens.
• pre-immune unvaccinated mouse bone marrow can be used to isolate antigen-specific naive B cells that bind to antigen-specific infectious agent reagents bearing broad neutralizing epitopes, the antibodies reflective of the naive B Cell Receptors (BCR) isolated and made recombinantly, and then immunogens selected for their high affinity binding to these BCR in order to discover immunogens that would selectively drive desired broad neutralizing B cell lineages to mature in peripheral lymph nodes and spleen.
• BCR naive B Cell Receptors
• VELOCIMMUNE® humanized mice can also be vaccinated with existing immunogens that themselves are antigenic for the broad neutralizing antibodies one is trying to induce, and the antibodies induced are then isolated by making monoclonal antibodies from the spleens or lymph nodes of the immunized mice, the antibodies made recombinantly, and then the most mature antibodies in the desired lineage used as a template to further drive the desired lineage to full expression of the desired function, e.g., a protective effector function against an infectious agent.
• That protective function can include virus neutralization, antibody-dependent cellular cytotoxicity against infectious agent-infected cells, prevention of movement of infectious agent across mucosal barriers, and blocking of entry of infectious agent in cell targets.
• the described processes can identify immunogens by iterative design based on using VELOCIMMUNE® humanized mouse-derived antibodies as templates for novel vaccine components.
• the best candidate system is the Regeneron VELOCIMMUNE® humanized mouse, which comprises a replacement at the endogenous mouse unrearranged heavy chain locus with the human unrearranged heavy chain locus; and a replacement of the endogenous mouse unrearranged ⁇ variable gene locus with the human unrearranged ⁇ light chain locus; wherein the human unrearranged loci are operably linked to endogenous mouse constant regions.
• the human unrearranged loci are operably linked to endogenous mouse constant regions.
• compositions and methods are provided for developing vaccine immunogens using animals that comprise humanized immunoglobulin loci.
• Non- human animals e.g., rodents, e.g., mice and rats
• rodents e.g., mice and rats
• human immunoglobulin variable domains and/or human unrearranged heavy chain variable gene segments and unrearranged light chain variable gene segments e.g. , , ⁇ , or ⁇ and ⁇
• suitable humanized non-human animals for generating B cells for use in the methods described herein include, for example, VELOCIMMUNE® humanized mice as described in, e.g. , US 20012/0322108A1 , US 2007/0061900A1 , US 201 1/0258710A1 , US 2001/0283376A1 , US Pat. No. 6,596,541 , US Pat. No. 7,105,248, each hereby incorporated by reference.
• non-human animals include, for example, non-human animals comprising a limited heavy chain repertoire are described in USSN 13.653,456, filed 17 October 2012; as well as non-human animals disclosed in USSN 61/658,466, filed 12 June 2012, and USSN 61/663,131 , filed 22 June 2012; each application and patent incorporated herein by reference.
• VELOCIMMUNE® humanized mice exhibit normal variable region usage and junctional diversity, norma! somatic hypermutation, normal numbers and distribution of B cells in spleen and lymph node, normal B cell differentiation in bone marrow, normal allelic exclusion, normal . ⁇ light chain ratios, normal serum levels for all Ig isotypes (IgM, lgG1 , lgG2a, lgG2b, lgG3, IgE, IgA), normal immune responses to target antigens, and normal ability to generate stable, wherein "normal” refers to identical wild-type mouse littermates.
• VELOCIMMMUNE® humanized mice are important for optimal B-cell-lineage immunogen design for vaccine development.
• VELOCIMMUNE® humanized mice (with restricted or non-restricted heavy chain variable loci) are outstanding platforms for B-cell-lineage immunogen design for vaccine development at least in part because they are an excellent source of bone marrow B cell precursors that reflect the naive human repertoire, a highly desired reagent for immunogen design (see, e.g., FIG.
• the VELOCIMMUNE® humanized mice (those used herein without a restricted repertoire) comprise a replacement of endogenous mouse V,D, and J gene segments with the full human repertoire of functional V, D, and J segments, operably linked to the mouse heavy chain locus.
• the VELOCIMMUNE® humanized mice (those used herein without a restricted repertoire) also comprise a replacement of endogenous mouse V and J gene segments with the full human repertoire of functional human VK and JK gene segments.
• Non-human Animals with Restricted Heavy Chain Repertoires V H 1 -69 and Beyond [000113] VELOCI MUNE® humanized mice with restricted repertoires, .e.g., the V H 1 -69 mouse used herein, comprises a replacement at the endogenous mouse locus of all functional mouse heavy chain V, D, and J segments with a single human V H 1-69 gene segment (present without polymorphs) operably linked to a plurality of human D segments and a plurality of human J segments, which are operably linked to an endogenous mouse immunoglobulin heavy chain locus.
• V H 1-69-derived heavy chains have been observed in a variety of antigen-specific antibody repertoires of therapeutic significance. For instance, V H 1-69 was frequently observed in heavy chain transcripts of an IgE repertoire of peripheral blood lymphocytes in young children with atopic disease (Bando et al.
• V H 1-69-derived heavy chains with a high degree of somatic hypermutation also occur in B cell lymphomas (Perez et al., (2009) Primary cutaneous B-cell lymphoma is associated with somatically hypermutated immunoglobulin variable genes and frequent use of V H 1-69 and V H 4-59 segments, British Journal of Dermatology 162:61 1-618), whereas some V H 1 -69-derived heavy chains with essentially germline sequences ⁇ i.e., little to no somatic hypermutation) have been observed among autoantibodies in patients with blood disorders (Pos ef al. (2008) V H 1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura, Journal of Thrombosis and Haemostasis 7:421-4
• V H 1-69-derived sequences (2000) Salivary gland mucosa-associated lymphoid tissue lymphoma immunoglobulin V H genes show frequent use of V H 1 -69 with distinctive CDR3 features, Blood 95(12):3878-3884; Kunert et al.
• V H 1-69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen, Blood 97(4): 1023-1026; Carbonari et al., (2005) Hepatitis C virus drives the unconstrained monoclonal expansion of V H 1-69-expressing memory B cells in type II cryoglobulinemia: A model of infection-driven lymphomagenesis, Journal of Immunology 74:6532-6539; Wang and Palese (2009) Universal epitopes of influenza virus hemagglutinins?, Nature Structural & Molecular Biology 16(3):233- 234; Sui et al.
• non-human animals that can be used as a platform for making B cell clones.
• Other humanized rodents can be used to generate B cell populations as well, including systems that employ fully human trangenes or chimeric human variable/non-human constant transgenes, whether the non-human animals be mice, rats, or other non-human animals, e.g., other rodents.
• suitable humanized non-human animals can comprise a full or a partial repertoire of heavy and/or light chain gene segments.
• all that is desired is the ability of the non-human animal to develop a B cell clone expressing a variable domain that is broadly neutralizing (or binds the pathogen of interest with sufficiently desirable characteristics, e.g., high avidity, high specificity, etc.) that can be further developed to exhibit desired characteristics.
• non-human animals e.g., rodents, e.g., mice or rats
• rodents e.g., mice or rats
• B cell lineage design methods described herein are useful in methods of vaccine design as against human pathogens, based on B cell lineage design methods described herein.
• the non-human animal e.g., a rodent, e.g., a mouse or rat
• a modified endogenous non-human immunoglobulin heavy chain locus comprising a replacement of all functional V gene segments with a single human V gene segment (or a single human V gene segment present in multiple polymorphic forms or copy number), wherein the non-human immunoglobulin heavy chain locus is incapable of rearrangement to form a heavy chain variable gene that is derived from a V gene segment other than the single human V gene segment (or one of the polymorphic forms or copies).
• the single human V gene segment is V H 1-69. In one embodiment, the single human V gene segment is V H 1-2.
• the locus comprises at least one human or non- human D H gene segment, and one human or non-human J H gene segment.
• the locus comprises a human DH gene segment and a human JH gene segment.
• the locus comprises a human J H gene segment.
• the locus comprises a human V H 1-69 gene segment (present as a single copy or multiple copies of different polymorphic variants), all functional human D H gene segments, and all functional human J H gene segments.
• the locus comprises a human V H 1-2 gene segment (present as a single copy or multiple copies of different polymorphic forms), all functional human DH gene segments, and all functional human J H gene segments.
• the human V, D, and J gene segments are operably linked to a mouse constant region gene at an endogenous mouse heavy chain locus.
• the mouse heavy chain locus comprises a wild-type repertoire of mouse immunoglobulin constant region sequences.
• a non-human animal comprising a modified immunoglobulin heavy chain locus
• a non-human animal comprising a modified immunoglobulin heavy chain locus
• the heavy chain V segment is a human segment.
• the one or more D segments are human D segments.
• the one or more J segments are human J segments.
• the one or more D segments and one or more J segments are human D and human J segments.
• the modified locus is a non-human locus.
• the non-human locus is modified with at least one human immunoglobulin sequence.
• the restriction is to one V segment family member.
• the one V segment family member is present in two or more copies.
• the one V segment family member is present as two or more variants ⁇ e.g., two or more polymorphic forms of the V segment family member).
• the one V segment is a human V segment family member.
• the one V segment family member is present in a number of variants as is observed in the human population with respect to that variant.
• the restriction is to a human V H 1-69 gene segment.
• the human V H 1-69 gene segment is present in two or more copies.
• the human V H 1-69 gene segment is present as two or more variants ⁇ e.g., two or more polymorphic forms the human V H 1 -69 gene).
• the human VH1-69 gene segment is present in a number of variants as is observed in the human population with respect to the human V H 1-69 gene segment.
• the restriction is to a human V H 1 -2 gene segment.
• the human V H 1-2 gene segment is present in two or more copies.
• the human V H 1-2 gene segment is present as two or more variants ⁇ e.g., two or more polymorphic forms the human V H 1-2 gene).
• the human V H 1-2 gene segment is present in a number of variants as is observed in the human population with respect to the human V H 1-2 gene segment.
• a non-human animal comprising a humanized heavy chain variable immunoglobulin locus that comprises a single functional human V segment.
• the single functional human V segment is selected from a V H 1-2, V H 1-3, V H 1-8, V H 1-18, V H 1-24, V H 1 -45, V H 1 -46, V H 1-58, V H 1 - 69, V H 2-5, V H 2-26, V H 2-70, V H 3-7, V H 3-9, V H 3-1 1 , V H 3-13, V H 3-15, V H 3-16, V H 3-20, V H 3-21 , V H 3-23, V H 3-30, V H 3-30-3, V H 3-30-5, V H 3-33, V H 3-35, V H 3-38, V H 3-43, V H 3- 48, V H 3-49, V H 3-53, V H 3-64, V H 3-66, V H 3-72, V H 3-73, V H 3-74, V H 4-4, V H H
• V segment is present in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or 13 polymorphic forms found in the human population.
• the single functional human V segment is a V H 1-2 segment; in a specific embodiment, the single functional human
• V segment is present in 1 , 2, 3, 4, or 5 polymorphic forms found in the human population.
• a non-human animal comprising a restricted immunoglobulin heavy chain V gene segment repertoire is provided, wherein the non-human animal further comprises one or more human immunoglobulin light chain variable segments (VK).
• VK human immunoglobulin light chain variable segments
• the one or more VK segments are operably linked to one or more human J segments.
• the J segments are human JK segments.
• the non- human animal does not express an immunoglobulin ⁇ light chain.
• the non-human animal does not comprise a functional human or functional endogenous immunoglobulin ⁇ light chain variable locus.
• the non-human animal is a rodent.
• the rodent is selected from a mouse and a rat.
• the non-human animal comprises a replacement at the endogenous non-human immunoglobulin VK locus of all or substantially all functional endogenous VK segments with one or more functional human VK segments.
• the replacement is with all or substantially all functional human immunoglobulin VK segments.
• the non-human animal comprises a replacement at the endogenous non-human immunoglobulin VK locus of all or substantially all functional endogenous VK gene segments with human VK gene segments selected from VK4-1 , VK5-2, VK7-3, VK2-4, VK1-5, VK1 -6, VK3-7, VK1-8, VK1-9, VK2-10, VK3- 1 1 , 1 -1 2, VK1 -1 3, VK2-14, VK3- 5, VK1 -16, VK1 -1 7, VK2-18, VK2-19, VK3-20, VK6-21 , VK1 -22, VK1 -23, VK2-24, VK3-25, VK2-26, VK1 -27, 2-28, VK2-29, VK2-30, VK3-31 , VK1 -32, VK1 -33, VK3-34, VK1 -35, VK2-36, VK1 -37,
• the non-human animal comprises a replacement at the endogenous non-human immunoglobulin JK locus of all or substantially all functional endogenous non-human immunoglobulin JK segments with one or more functional human immunoglobulin JK segments.
• the replacement is with all or substantially all functional human immunoglobulin JK segments.
• the non-human animal comprises a replacement at the endogenous non-human immunoglobulin JK locus of all or substantially all functional endogenous non-human immunoglobulin JK gene segments with human JK gene segments selected from JK1 , JK2, JK3, J 4, JK5, and a combination thereof.
• the non-human animal comprises an immunoglobulin heavy chain variable region locus that comprises a repertoire of V segments consisting essentially of a single V segment and/or polymorphic variants thereof.
• the single immunoglobulin heavy chain V segment is a human V H 1 -69 segment
• the non-human animal further comprises a replacement of all functional non-human D H segments with all functional human D H segments, and further comprises a replacement of all functional non-human J H segments with all functional human J H segments
• the immunoglobulin heavy chain variable region locus is operably linked to a human or non-human constant region gene sequence.
• the constant region gene sequence is an endogenous non-human constant region gene sequence.
• the non-human animal rearranges segments at the non-human immunoglobulin heavy chain locus to form a gene encoding heavy chain variable region comprising a human V H 1-69 sequence, a human D H sequence, a human J H sequence, and a mouse constant region sequence.
• the non-human animal comprises an immunoglobulin heavy chain variable region locus that comprises a repertoire of V segments consisting essentially of a single V segment and/or polymorphic variants thereof.
• the single immunoglobulin heavy chain V segment is a human V H 1 -2 segment
• the non-human animal further comprises a replacement of all functional non-human D H segments with all functional human D H segments, and further comprises a replacement of all functional non-human J H segments with all functional human J H segments
• the immunoglobulin heavy chain variable region locus is operably linked to a human or non-human constant region gene sequence.
• the constant region gene sequence is an endogenous non-human constant region gene sequence.
• the non-human animal rearranges segments at the non-human immunoglobulin heavy chain locus to form a gene encoding heavy chain variable region comprising a human V H 1-2 sequence, a human DH sequence, a human J H sequence, and a mouse constant region sequence.
• a non-human animal comprising a restricted immunoglobulin heavy chain V segment repertoire
• the non- human animal comprises one or more human ⁇ light chain variable (VX) segments.
• the one or more human VX segments are operably linked to one or more human J segments.
• the J segments are human JX segments.
• the non-human animal does not express a ⁇ light chain.
• the non-human animal does not comprise a functional human or non-human ⁇ light chain variable locus.
• the non-human animal further comprises a replacement of all or substantially all functional non-human immunoglobulin
• the non-human animal comprises a replacement of all or substantially all functional non-human immunoglobulin VX segments with one or more functional human immunoglobulin VX segments.
• the replacement is with all or substantially all functional human immunoglobulin VX segments.
• the non-human animal comprises a replacement of all or substantially all functional non-human VX segments with a fragment of cluster A of the human X light chain locus.
• the fragment of cluster A of the human X light chain locus comprises human VX gene segments VX3- 27 through VX3-1.
• the non-human animal comprises a replacement of all or substantially all functional non-human VX segments with a fragment of cluster B of the human X light chain locus.
• the fragment of cluster B of the human X light chain locus comprises human VX gene segments VX5- 52 through VX1 -40.
• the non-human animal comprises a replacement of all or substantially all functional non-human VX segments with a fragment of cluster A and a fragment of cluster B of the human X light chain locus, wherein as a result of the replacement comprise human VX gene segments VX5-52 through VX3-1.
• the non-human animal comprises a replacement of all or substantially all functional non-human VX segments with at least 12 human VX gene segments, at least 28 human VX gene segments, or at least 40 human VX gene segments.
• the non-human animal comprises a replacement of all or substantially all functional non-human immunoglobulin ⁇ gene segments with one or more functional human immunoglobulin ⁇ gene segments.
• the replacement is with all or substantially all functional human immunoglobulin J gene segments.
• the functional human ⁇ gene segments include ⁇ 1 , J 2, ⁇ 3 and ⁇ 7.
• the non-human animal comprises an immunoglobulin heavy chain variable (V H ) region locus that comprises only a single VH segment, wherein the single V H segment is a human V H 1 -69 segment or a human V H 1-2 segment, and further comprises a replacement of all functional non-human D H segments with all functional human D H segments, and further comprises a replacement of all functional non-human J H segments with all functional human J H segments, and wherein the V H region locus is operably linked to a human or non- human constant region gene sequence.
• the constant region gene sequence is a non-human constant region gene sequence, e.g., an endogenous non-human constant gene sequence.
• the non-human animal rearranges segments at the non-human immunoglobulin heavy chain locus to form a gene encoding an immunoglobulin heavy chain variable region comprising a human V H 1 -69 sequence (or a human V H 1-2 sequence), a human D H sequence, a human J H sequence, and an endogenous non-human constant region sequence.

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